title: Propidium monoazide combined with real-time quantitative PCR to quantify viable Alternaria spp. contamination in tomato products.
abstract: Alternaria is a common contaminating genus of fungi in fruits, grains, and vegetables that causes severe economic losses to farmers and the food industry. Furthermore, it is claimed that Alternaria spp. are able to produce phytotoxic metabolites, and mycotoxins that are unsafe for human and animal health. DNA amplification techniques are being increasingly applied to detect, identify, and quantify mycotoxigenic fungi in foodstuffs, but the inability of these methods to distinguish between viable and nonviable cells might lead to an overestimation of mycotoxin-producing living cells. A promising technique to overcome this problem is the pre-treatment of samples with nucleic acid intercalating dyes, such as propidium monoazide (PMA), prior to quantitative PCR (qPCR). PMA selectively penetrates cells with a damaged membrane inhibiting DNA amplification during qPCRs. In our study, a primer pair (Alt4-Alt5) to specifically amplify and quantify Alternaria spp. by qPCR was designed. Quantification data of qPCR achieved a detection limit of 10(2)conidia/g of tomato. Here, we have optimized for the first time a DNA amplification-based PMA sample pre-treatment protocol for detecting viable Alternaria spp. cells. Artificially inoculated tomato samples treated with 65μM of PMA, showed a reduction in the signal by almost 7cycles in qPCR between live and heat-killed Alternaria spp. conidia. The tomato matrix had a protective effect on the cells against PMA toxicity, reducing the efficiency to distinguish between viable and nonviable cells. The results reported here indicate that the PMA-qPCR method is a suitable tool for quantifying viable Alternaria cells, which could be useful for estimating potential risks of mycotoxin contamination.
authors.affiliation: Applied Mycology Unit, Food Technology Department, University of Lleida, UTPV-XaRTA, Agrotecnio Center, Av. Rovira Roure 191, 25198 Lleida, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Crespo-Sempere
authors.forename: Núria
authors.initials: N
authors.lastname: Estiarte
authors.forename: Sonia
authors.initials: S
authors.lastname: Marín
authors.forename: Vicente
authors.initials: V
authors.lastname: Sanchis
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Ramos
mesh_terms: D000528:Alternaria / Q000235:genetics* / Q000302:isolation & purification; D001386:Azides / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004271:DNA, Fungal / Q000378:metabolism; D005516:Food Microbiology / Q000379:methods*; D018551:Solanum lycopersicum / Q000382:microbiology*; D050296:Microbial Viability; D011419:Propidium / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D001386:Azides; D017931:DNA Primers; D004271:DNA, Fungal; C533957:propidium monoazide; D011419:Propidium
keywords: Alternaria spp.; Mycotoxins; Propidium monoazide; Quantitative polymerase chain reaction; Tomato